Authors
Sergé Arnauld, Bertaux Nicolas, Rigneault Hervé, and Marguet Didier
Summary
Single fluorescence molecule microscopy provides a privileged approach achieving a spatial resolution close to the molecular scale1-5. The position of an isolated fluorescent molecule is accurately determined by the center of the signal. The counterpart is a necessity for imaging sparse fluorescent molecules. Consequently, single particle tracking (SPT) is only amenable when handling a very limited number of targets at the same time. This major drawback has been partly circumvented by the development of super-resolution optical imaging techniques6. Among them, photoactivation localization microscopy (PALM7, FPALM8) or stochastic optical reconstruction microscopy (STORM9) permit the localization of thousands of single fluorophores per acquisition. This is experimentally performed by sequentially turning on a fraction of the fluorescently-labeled molecules. The combination of PALM with live-cell SPT (sptPALM10) allows to simultaneously track a large number of molecules. This approach provides significant insight into molecular dynamics and interactions taking place at the cell membrane. Here, we detail a tracking approach, namely multiple-target tracing (MTT), optimized for large numbers of targets, allowing highly resolved spatiotemporal investigations on molecular dynamics at the cell surface. Use of dedicated signal detection tools11,12 allows efficiently detecting and estimating particles, then reconnecting trajectories over time, with a controlled and optimized accuracy.
Further details
The protocol was published on Protocol Exchange in 2008. To see the entire protocol, click on the source link.
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